Methods for detecting vitamin D metabolites by mass spectrometry

ABSTRACT

Provided are methods of detecting the presence or amount of a vitamin D metabolite in a sample using mass spectrometry. The methods generally directed to ionizing a vitamin D metabolite in a sample and detecting the amount of the ion to determine the presence or amount of the vitamin D metabolite in the sample. Also provided are methods to detect the presence or amount of two or more vitamin D metabolites in a single assay.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/848,325, filed Dec. 20, 2017, now U.S. Pat. No. 10,267,810, which isa continuation of U.S. application Ser. No. 15/388,844, filed Dec. 22,2016, now U.S. Pat. No. 9,880,180, which is a continuation of U.S.application Ser. No. 15/005,801, filed Jan. 25, 2016, now U.S. Pat. No.9,529,004, which is a continuation of U.S. application Ser. No.14/180,722, filed Feb. 14, 2014, now U.S. Pat. No. 9,244,084, which is acontinuation of U.S. application Ser. No. 13/871,457, now U.S. Pat. No.8,936,943, filed Apr. 26, 2013, which is a continuation of U.S.application Ser. No. 13/327,650, now U.S. Pat. No. 8,431,411, filed Dec.15, 2011, which is a continuation of U.S. application Ser. No.13/115,916, now U.S. Pat. No. 8,101,427, filed May 25, 2011, which is acontinuation of U.S. application Ser. No. 11/386,215, now U.S. Pat. No.7,972,867, filed Mar. 21, 2006, which is a continuation-in-part of U.S.application Ser. No. 11/101,166, now U.S. Pat. No. 7,745,226, filed Apr.6, 2005, each of which is incorporated by reference in its entiretyherein.

TECHNOLOGY FIELD

The invention relates to the detection of vitamin D metabolites. In aparticular aspect, the invention relates to methods for detectingvitamin D metabolites by mass spectrometry.

BACKGROUND

Vitamin D is an essential nutrient with important physiological roles inthe positive regulation of calcium (Ca²⁺) homeostasis. Vitamin D can bemade de novo in the skin by exposure to sunlight or it can be absorbedfrom the diet. There are two forms of vitamin D; vitamin D₂(ergocalciferol) and vitamin D₃ (cholecalciferol). Vitamin D₃ is theform synthesized de novo by animals. It is also a common supplementadded to milk products and certain food products produced in the UnitedStates. Both dietary and intrinsically synthesized vitamin D₃ mustundergo metabolic activation to generate bioactive metabolites. Inhumans, the initial step of vitamin D₃ activation occurs primarily inthe liver and involves hydroxylation to form the intermediate metabolite25-hydroxyvitamin D₃ (25-hydroxycholecalciferol; calcifediol; 25OHD₃).Calcifediol is the major form of vitamin D₃ in the circulation.Circulating 25OHD₃ is then converted by the kidney to1,25-dihydroxyvitamin D₃ (calcitriol; 1,25(OH)₂D₃), which is generallybelieved to be the metabolite of vitamin D₃ with the highest biologicalactivity.

Vitamin D₂ is derived from fungal and plant sources. Manyover-the-counter dietary supplements contain ergocalciferol (vitamin D₂)rather than cholecalciferol (vitamin D₃). Drisdol, the only high-potencyprescription form of vitamin D available in the United States, isformulated with ergocalciferol. Vitamin D₂ undergoes a similar pathwayof metabolic activation in humans as vitamin D₃, forming the metabolites25-hydroxyvitamin D₂ (25OHD₂) and 1,25-dihydroxyvitamin D₃(1,25(OH)₂D₂). Vitamin D₂ and vitamin D₃ have long been assumed to bebiologically equivalent in humans, however recent reports suggest thatthere may be differences in the bioactivity and bioavailability of thesetwo forms of vitamin D (Armas et. al., (2004) J. Clin. Endocrinol.Metab. 89:5387-5391).

Measurement of vitamin D, the inactive vitamin D precursor, is rare inclinical settings and has little diagnostic value. Rather, serum levelsof 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂ (total25-hydroxyvitamin D; “25OHD”) are a useful index of vitamin Dnutritional status and the efficacy of certain vitamin D analogs.Therefore, the measurement of 25OHD is commonly used in the diagnosisand management of disorders of calcium metabolism. In this respect, lowlevels of 25OHD are indicative of vitamin D deficiency associated withdiseases such as hypocalcemia, hypophosphatemia, secondaryhyperparathyroidism, elevated alkaline phosphatase, osteomalacia inadults and rickets in children. In patients suspected of vitamin Dintoxication, elevated levels of 25OHD distinguishes this disorder fromother disorders that cause hypercalcemia.

Measurement of 1,25(OH)₂D is also used in clinical settings, however,this metabolite has a more limited diagnostic usefulness than 25OHD.Factors that contribute to limitations of the diagnostic values of1,25(OH)₂D as an index of vitamin D status include the precision of theendogenous regulation of renal production of the metabolite and itsshort half-life in circulation. However, certain disease states such askidney failure can be diagnosed by reduced levels of circulating1,25(OH)₂D and elevated levels of 1,25(OH)₂D may be indicative of excessparathyroid hormone or may be indicative of certain diseases such assarcoidosis or certain types of lymphoma.

Detection of vitamin D metabolites has been accomplished byradioimmunoassay with antibodies co-specific for 25-hydroxyvitamin D₃and 25-hydroxyvitamin D₂. Because the current immunologically-basedassays do not separately resolve 25-hydroxyvitamin D₃ and25-hydroxyvitamin D₂, the source of a deficiency in vitamin D nutritioncannot be determined without resorting to other tests. More recently,reports have been published that disclose methods for detecting specificvitamin D metabolites using mass spectrometry. For example Yeung B, etal., J Chromatogr. 1993, 645(1):115-23; Higashi T, et al., Steroids.2000, 65(5):281-94; Higashi T, et al., Biol Pharm Bull. 2001,24(7):738-43; and Higashi T, et al., J Pharm Biomed Anal. 2002,29(5):947-55 disclose methods for detecting various vitamin Dmetabolites using liquid chromatography and mass spectrometry. Thesemethods require that the metabolites be derivatized prior to detectionby mass-spectrometry. Methods to detect underivatized 1,25(OH)₂D₃ byliquid chromatography/mass-spectrometry are disclosed in Kissmeyer andSonne, J Chromatogr A. 2001, 935(1-2):93-103.

SUMMARY

The present invention provides methods for detecting the presence oramount of a vitamin D metabolite in a sample by mass spectrometry,including tandem mass spectrometry. Preferably, the methods of theinvention do not include derivatizing the vitamin D metabolites prior tothe mass spectrometry analysis.

In one aspect, the invention provides a method for determining thepresence or amount of a vitamin D metabolite in a sample. The method mayinclude: (a) ionizing the vitamin D metabolite, if present in thesample; and (b) detecting the presence or amount of the ion by massspectrometry and relating presence or amount of the detected ion to thepresence or amount of the vitamin D metabolite in the sample. In somepreferred embodiments, the ionization step (a) may include (i) ionizingthe vitamin D metabolite, if present in the sample, to produce an ion ofthe vitamin D metabolite; (ii) isolating the ion of step (i) by massspectrometry to provide a precursor ion; and (iii) effecting a collisionbetween the precursor ion and an inert collision gas to produce at leastone fragment ion detectable in a mass spectrometer. Preferably, at leastone of the fragment ions is specific for the vitamin D metabolite ofinterest. In certain embodiments of the invention, the fragment ions tobe detected include at least one fragment ion other than that whichresults solely by a dehydration or deamination of the precursor ion. Insome particularly preferred embodiments, the precursor ion is aprotonated and dehydrated ion of the vitamin D metabolite. In certainembodiments the vitamin D metabolite is one or more vitamin Dmetabolites selected from the group consisting of 25-hydroxyvitamin D₃;25-hydroxyvitamin D₂; 1,25-dihydroxyvitamin D₂; and1,25-dihydroxyvitamin D₃.

In a related aspect, the invention provides a method for determining thepresence or amount of a vitamin D metabolite in a sample by tandem massspectrometry. The method may involve (a) generating a protonated anddehydrated precursor ion of the vitamin D metabolite if present in thesample; (b) generating one or more fragment ions of the precursor ion;and (c) detecting the presence or amount of one or more of the ionsgenerated in step (a) or (b) or both and relating the detected ions tothe presence or amount of the vitamin D metabolite in the sample. Incertain embodiments, the method is used to detect the presence or amountof two or more vitamin D metabolites in a single assay. Preferably, themethod does not involve derivatizing the samples or the vitamin Dmetabolites prior to analysis by mass spectrometry. In certainembodiments the vitamin D metabolite is one or more vitamin Dmetabolites selected from the group consisting of 25-hydroxyvitamin D₃;25-hydroxyvitamin D₂; 1,25-dihydroxyvitamin D₂; and1,25-dihydroxyvitamin D₃.

In another aspect the invention provides a method for determining thepresence or amount of two or more vitamin D metabolites in a sample in asingle assay. The method includes ionizing the vitamin D metabolites, ifpresent in the sample, to generate ions specific for each of the vitaminD metabolites of interest, detecting the presence or amount of the ionsby mass spectrometry, and relating the presence or amount of the ions tothe presence or amount of the vitamin D metabolites in the sample. Incertain embodiments the mass spectrometry analysis of the method istandem mass spectrometry.

As used herein, the term “vitamin D metabolite” refers to any chemicalspecies that may be found in the circulation of an animal which isformed by a biosynthetic or metabolic pathway for vitamin D or asynthetic vitamin D analog. Vitamin D metabolites include forms ofvitamin D that are generated by a biological organism, such as ananimal, or that are generated by biotransformation of a naturallyoccurring form of vitamin D or a synthetic vitamin D analog. In certainpreferred embodiments, a vitamin D metabolite is formed by thebiotransformation of vitamin D₂ or vitamin D₃. In particularly preferredembodiments, the vitamin D metabolite is one or more compounds selectedfrom the group consisting of 25-hydroxyvitamin D₃, 25-hydroxyvitamin D₂,1,25-dihydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₂.

As used herein, the term “purification” refers to a procedure thatenriches the amount of one or more analytes of interest relative to oneor more other components of the sample. Purification, as used hereindoes not require the isolation of an analyte from all others. Inpreferred embodiments, a purification step or procedure can be used toremove one or more interfering substances, e.g., one or more substancesthat would interfere with the operation of the instruments used in themethods or substances that may interfere with the detection of ananalyte ion by mass spectrometry.

As used herein, “biological sample” refers to any sample from abiological source. As used herein, “body fluid” means any fluid that canbe isolated from the body of an individual. For example, “body fluid”may include blood, plasma, serum, bile, saliva, urine, tears,perspiration, and the like.

As used herein, “derivatizing” means reacting two molecules to form anew molecule. Derivatizing agents may include isothiocyanate groups,dinitro-fluorophenyl groups, nitrophenoxycarbonyl groups, and/orphthalaldehyde groups.

As used herein, “chromatography” refers to a process in which a chemicalmixture carried by a liquid or gas is separated into components as aresult of differential distribution of the chemical entities as theyflow around or over a stationary liquid or solid phase.

As used herein, “liquid chromatography” (LC) means a process ofselective retardation of one or more components of a fluid solution asthe fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). “Liquid chromatography”includes reverse phase liquid chromatography (RPLC), high performanceliquid chromatography (HPLC) and high turbulence liquid chromatography(HTLC).

As used herein, the term “HPLC” or “high performance liquidchromatography” refers to liquid chromatography in which the degree ofseparation is increased by forcing the mobile phase under pressurethrough a stationary phase, typically a densely packed column.

As used herein, the term “gas chromatography” refers to chromatographyin which the sample mixture is vaporized and injected into a stream ofcarrier gas (as nitrogen or helium) moving through a column containing astationary phase composed of a liquid or a particulate solid and isseparated into its component compounds according to the affinity of thecompounds for the stationary phase

As used herein, “mass spectrometry” (MS) refers to an analyticaltechnique to identify compounds by their mass. MS technology generallyincludes (1) ionizing the compounds to form charged compounds; and (2)detecting the molecular weight of the charged compound and calculating amass-to-charge ratio (m/z). The compound may be ionized and detected byany suitable means. A “mass spectrometer” generally includes an ionizerand an ion detector. See, e.g., U.S. Pat. No. 6,204,500, entitled “MassSpectrometry From Surfaces;” U.S. Pat. No. 6,107,623, entitled “Methodsand Apparatus for Tandem Mass Spectrometry;” U.S. Pat. No. 6,268,144,entitled “DNA Diagnostics Based On Mass Spectrometry;” U.S. Pat. No.6,124,137, entitled “Surface-Enhanced Photolabile Attachment And ReleaseFor Desorption And Detection Of Analytes;” Wright et al., ProstateCancer and Prostatic Diseases 2:264-76 (1999); and Merchant andWeinberger, Electrophoresis 21:1164-67 (2000).

The term “electron ionization” as used herein refers to methods in whichan analyte of interest in a gaseous or vapor phase interacts with a flowof electrons. Impact of the electrons with the analyte produces analyteions, which may then be subjected to a mass spectrometry technique.

The term “chemical ionization” as used herein refers to methods in whicha reagent gas (e.g. ammonia) is subjected to electron impact, andanalyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

The term “fast atom bombardment” as used herein refers to methods inwhich a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine.

The term “field desorption” as used herein refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

The term “ionization” as used herein refers to the process of generatingan analyte ion having a net electrical charge equal to one or moreelectron units. Negative ions are those having a net negative charge ofone or more electron units, while positive ions are those having a netpositive charge of one or more electron units.

The term “operating in negative ion mode” refers to those massspectrometry methods where negative ions are detected. Similarly,“operating in positive ion mode” refers to those mass spectrometrymethods where positive ions are detected.

The term “desorption” as used herein refers to the removal of an analytefrom a surface and/or the entry of an analyte into a gaseous phase.

The term “about” as used herein in reference to quantitativemeasurements, refers to the indicated value plus or minus 10%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the linearity of the quantification of 25OHD₂ in seriallydiluted stock samples using an LC-MS/MS assay. Details are described inExample 4.

FIG. 2 shows the linearity of the quantification of 25OHD₃ in seriallydiluted stock samples using an LC-MS/MS assay. Details are described inExample 4.

FIG. 3 shows the linearity of the quantification by LC-MS/MS of seriallydiluted samples containing 25OHD₂ and 25OHD₃ to final concentrations of512 ng/mL. Details are described in Example 4.

FIG. 4 shows the correlation between detection of total25-hydroxyvitamin D using an LC-MS/MS assay and a commercially availableradioimmunoassay kit. Details are described in Example 6.

DETAILED DESCRIPTION

Disclosed are methods for detecting the presence or amount of one ormore vitamin D metabolites in a sample. In certain aspects the methodinvolves ionizing the vitamin D metabolite(s), detecting the ion(s) bymass spectrometry, and relating the presence or amount of the ion(s) tothe presence or amount of the vitamin D metabolite(s) in the sample. Themethod may include (a) purifying a vitamin D metabolite, if present inthe sample, (b) ionizing the purified vitamin D metabolite and (c)detecting the presence or amount of the ion, wherein the presence oramount of the ion is related to the presence or amount of the vitamin Dmetabolite in the sample. In preferred embodiments, the ionizing step(b) may include (i) ionizing a vitamin D metabolite, if present in thesample, to produce an ion; (ii) isolating the vitamin D metabolite ionby mass spectrometry to provide a precursor ion; and (iii) effecting acollision between the isolated precursor ion and an inert collision gasto produce at least one fragment ion detectable in a mass spectrometer.In certain preferred embodiments the precursor ion is a protonated anddehydrated ion of the vitamin D metabolite.

In a related aspect, the invention provides a method for determining thepresence or amount of a vitamin D metabolite in a test sample by tandemmass spectrometry. The method may involve (a) generating a protonatedand dehydrated precursor ion of the vitamin D metabolite; (b) generatingone or more fragment ions of the precursor ion; and (c) detecting thepresence or amount of one or more of the ions generated in step (a) or(b) or both and relating the detected ions to the presence or amount ofsaid vitamin D metabolite in the sample.

In certain preferred embodiments of the invention, at least one fragmention is detected, wherein the presence or amount of the precursor and/orat least one fragment ion is related to the presence or amount of thevitamin D metabolite in the sample. Preferably at least one fragment ionis specific for the vitamin D metabolite of interest. In someembodiments, the methods of the invention can be used to detect andquantify two or more vitamin D metabolites in a single assay. In certainembodiments, the vitamin D metabolite is one or more vitamin Dmetabolites selected from the group consisting of 25-hydroxyvitamin D₃;25-hydroxyvitamin D₂; 1,25-dihydroxyvitamin D₂; and1,25-dihydroxyvitamin D₃.

Suitable samples include any sample that might contain the analyte ofinterest and/or one or more metabolites or precursors thereof. Forexample, samples obtained during the manufacture of an analyte can beanalyzed to determine the composition and yield of the manufacturingprocess. In certain embodiments, a sample is a biological sample; thatis, a sample obtained from any biological source, such as an animal, acell culture, an organ culture, etc. Particularly preferred are samplesobtained from a human, such as a blood, plasma, serum, hair, muscle,urine, saliva, tear, cerebrospinal fluid, or other tissue sample. Suchsamples may be obtained, for example, from a patient seeking diagnosis,prognosis, or treatment of a disease or condition. The vitamin Dmetabolites may be derivatized prior to mass spectrometry, however, incertain preferred embodiments, sample preparation excludes the use ofderivatization.

Samples may be processed or purified to obtain preparations that aresuitable for analysis by mass spectrometry. Such purification willusually include chromatography, such as liquid chromatography, and mayalso often involve an additional purification procedure that isperformed prior to chromatography. Various procedures may be used forthis purpose depending on the type of sample or the type ofchromatography. Examples include filtration, extraction, precipitation,centrifugation, dilution, combinations thereof and the like. Proteinprecipitation is one preferred method of preparing a liquid biologicalsample, such as serum or plasma, for chromatography. Such proteinpurification methods are well known in the art, for example, Polson etal., Journal of Chromatography B 785:263-275 (2003), describes proteinprecipitation methods suitable for use in the methods of the invention.Protein precipitation may be used to remove most of the protein from thesample leaving vitamin D metabolites soluble in the supernatant. Thesamples can be centrifuged to separate the liquid supernatant from theprecipitated proteins. The resultant supernatant can then be applied toliquid chromatography and subsequent mass spectrometry analysis. In oneembodiment of the invention, the protein precipitation involves addingone volume of the liquid sample (e.g. plasma) to about four volumes ofmethanol. In certain embodiments, the use of protein precipitationobviates the need for high turbulence liquid chromatography (“HTLC”) oron-line extraction prior to HPLC and mass spectrometry. Accordingly insuch embodiments, the method involves (1) performing a proteinprecipitation of the sample of interest; and (2) loading the supernatantdirectly onto the HPLC-mass spectrometer without using on-lineextraction or high turbulence liquid chromatography (“HTLC”).

The purification step may include chromatography, preferably liquidchromatography, more preferably high performance liquid chromatography(HPLC). In some preferred embodiments the chromatography is not gaschromatography. Preferably, the methods of the invention are performedwithout subjecting the samples, or the vitamin D metabolites ofinterest, to gas chromatography prior to mass spectrometric analysis.

Various methods have been described involving the use of HPLC for sampleclean-up prior to mass spectrometry analysis. See, e.g., Taylor et al.,Therapeutic Drug Monitoring 22:608-12 (2000) (manual precipitation ofblood samples, followed by manual C18 solid phase extraction, injectioninto an HPLC for chromatography on a C18 analytical column, and MS/MSanalysis); and Salm et al., Clin. Therapeutics 22 Supl. B:B71-B85 (2000)(manual precipitation of blood samples, followed by manual C18 solidphase extraction, injection into an HPLC for chromatography on a C18analytical column, and MS/MS analysis). One of skill in the art canselect HPLC instruments and columns that are suitable for use in theinvention. The chromatographic column typically includes a medium (i.e.,a packing material) to facilitate separation of chemical moieties (i.e.,fractionation). The medium may include minute particles. The particlesinclude a bonded surface that interacts with the various chemicalmoieties to facilitate separation of the chemical moieties such asvitamin D metabolites. One suitable bonded surface is a hydrophobicbonded surface such as an alkyl bonded surface. Alkyl bonded surfacesmay include C-4, C-8, or C-18 bonded alkyl groups, preferably C-18bonded groups. The chromatographic column includes an inlet port forreceiving a sample and an outlet port for discharging an effluent thatincludes the fractionated sample. In the method, the sample (orpre-purified sample) is applied to the column at the inlet port, elutedwith a solvent or solvent mixture, and discharged at the outlet port.Different solvent modes may be selected for eluting the analytes ofinterest. For example, liquid chromatography may be performed using agradient mode, an isocratic mode, or a polytyptic (i.e. mixed) mode. Inpreferred embodiments, HPLC is performed on a multiplexed analyticalHPLC system with a C18 solid phase using isocratic separation with 100%methanol as the mobile phase.

Recently, high turbulence liquid chromatography (“HTLC”), also calledhigh throughput liquid chromatography, has been applied for samplepreparation prior to analysis by mass spectrometry. See, e.g., Zimmer etal., J. Chromatogr. A 854:23-35 (1999); see also, U.S. Pat. Nos.5,968,367; 5,919,368; 5,795,469; and 5,772,874. Traditional HPLCanalysis relies on column packings in which laminar flow of the samplethrough the column is the basis for separation of the analyte ofinterest from the sample. The skilled artisan will understand thatseparation in such columns is a diffusional process. In contrast, it isbelieved that turbulent flow, such as that provided by HTLC columns andmethods, may enhance the rate of mass transfer, improving the separationcharacteristics provided. In some embodiments, high turbulence liquidchromatography (HTLC), alone or in combination with one or morepurification methods, may be used to purify the vitamin D metabolite ofinterest prior to mass spectrometry. In such embodiments samples may beextracted using an HTLC extraction cartridge which captures the analyte,then eluted and chromatographed on a second HTLC column or onto ananalytical HPLC column prior to ionization. Because the steps involvedin these chromatography procedures can be linked in an automatedfashion, the requirement for operator involvement during thepurification of the analyte can be minimized. In certain embodiments ofthe method, samples are subjected to protein precipitation as describedabove prior to loading on the HTLC column; in alternative embodiments,the samples may be loaded directly onto the HTLC without being subjectedto protein precipitation.

Recently, research has shown that epimerization of the hydroxyl group ofthe A-ring of vitamin D₃ metabolites is an important aspect of vitaminD₃ metabolism and bioactivation, and that depending on the cell typesinvolved, 3-C epimers of vitamin D₃ metabolites (e.g., 3-epi-25(OH)D₃;3-epi-24,25(OH)₂D₃; and 3-epi-1,25(OH)₂D₃) are often major metabolicproducts. See Kamao et al., J. Biol. Chem., 279:15897-15907 (2004).Kamao et al., further provides methods of separating various vitamin Dmetabolites, including 3-C epimers, using Chiral HPLC. Accordingly, theinvention also provides methods of detecting the presence, absenceand/or amount of a specific epimer of one or more vitamin D metabolites,preferably vitamin D₃ metabolites, in a sample by (1) separating one ormore specific vitamin D metabolites by chiral chromatography, preferablychiral HPLC; and (2) detecting the presence and/or amount of one or morevitamin D metabolites using mass spectrometry methods as describedherein. The chiral chromatography procedures described in Kamao et al.,are suitable for the methods of the invention, however, one of ordinaryskill in the art understands that there are numerous other chiralchromatography methods that would also be suitable. In preferredembodiments the method includes, separating 25(OH)D₃ from3-epi-25(OH)D₃, if present in a sample, using chiral chromatography; anddetecting the presence and/or amount of the 25(OH)D₃ and the3-epi-25(OH)D₃ in the sample using mass spectrometry. In relatedembodiments, the method includes separating 1α,25(OH)₂D₃ from3-epi-1α,25(OH)₂D₃, if present in a sample, using chiral chromatography;and detecting the presence and/or amount of the 1α,25(OH)₂D₃ and the3-epi-1α,25(OH)₂D₃ in the sample using mass spectrometry. In certainembodiments of the invention, chiral chromatography is used inconjunction with the HTLC methods described above.

Mass spectrometry is performed using a mass spectrometer which includesan ion source for ionizing the fractionated sample and creating chargedmolecules for further analysis. For example ionization of the sample maybe performed by electrospray ionization (ESI), atmospheric pressurechemical ionization (APCI), photoinonization, electron ionization, fastatom bombardment (FAB)/liquid secondary ionization (LSIMS), matrixassisted laser desorption ionization (MALDI), field ionization, fielddesorption, thermospray/plasmaspray ionization, and particle beamionization. The skilled artisan will understand that the choice ofionization method can be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio (i.e., m/z). Suitable analyzers for determiningmass-to-charge ratios include quadropole analyzers, ion traps analyzers,and time-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected (i.e., usinga selective ion monitoring mode (SIM)), or alternatively, ions may bedetected using a scanning mode, e.g., multiple reaction monitoring (MRM)or selected reaction monitoring (SRM). Preferably, the mass-to-chargeratio is determined using a quadropole analyzer. For example, in a“quadrupole” or “quadrupole ion trap” instrument, ions in an oscillatingradio frequency field experience a force proportional to the DCpotential applied between electrodes, the amplitude of the RF signal,and m/z. The voltage and amplitude can be selected so that only ionshaving a particular m/z travel the length of the quadrupole, while allother ions are deflected. Thus, quadrupole instruments can act as both a“mass filter” and as a “mass detector” for the ions injected into theinstrument.

One can often enhance the resolution of the MS technique by employing“tandem mass spectrometry,” or “MS/MS.” In this technique, a precursorion (also called a parent ion) generated from a molecule of interest canbe filtered in an MS instrument, and the precursor ion is subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collision withatoms of an inert gas to produce the daughter ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquecan provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation can be used to eliminateinterfering substances, and can be particularly useful in complexsamples, such as biological samples.

Additionally, recent advances in technology, such as matrix-assistedlaser desorption ionization coupled with time-of-flight analyzers(“MALDI-TOF”) permit the analysis of analytes at femtomole levels invery short ion pulses. Mass spectrometers that combine time-of-flightanalyzers with tandem MS are also well known to the artisan.Additionally, multiple mass spectrometry steps can be combined inmethods known as “MS/MS^(n).” Various other combinations may beemployed, such as MS/MS/TOF, MALDI/MS/MS/TOF, or SELDI/MS/MS/TOF massspectrometry.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular m/z over agiven range (e.g., 100 to 1000 amu). The results of an analyte assay,that is, a mass spectrum, can be related to the amount of the analyte inthe original sample by numerous methods known in the art. For example,given that sampling and analysis parameters are carefully controlled,the relative abundance of a given ion can be compared to a table thatconverts that relative abundance to an absolute amount of the originalmolecule. Alternatively, molecular standards can be run with thesamples, and a standard curve constructed based on ions generated fromthose standards. Using such a standard curve, the relative abundance ofa given ion can be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity of the vitaminD metabolite. Methods of generating and using such standard curves arewell known in the art and one of ordinary skill is capable of selectingan appropriate internal standard. For example, an isotope of a vitamin Dmetabolite may be used as an internal standard, in preferred embodimentsthe vitamin D metabolite is a deuterated vitamin D metabolite, forexample ⁶D-25OHD₃. Numerous other methods for relating the presence oramount of an ion to the presence or amount of the original molecule willbe well known to those of ordinary skill in the art.

One or more steps of the methods of the invention can be performed usingautomated machines. In certain embodiments, one or more purificationsteps are performed on line, and more preferably all of the purificationand mass spectrometry steps may be performed in an on-line fashion.

In particularly preferred embodiments vitamin D metabolites are detectedand/or quantified using LC-MS/MS as follows. The samples are subjectedto liquid chromatography, preferably HPLC, the flow of liquid solventfrom the chromatographic column enters the heated nebulizer interface ofa LC-MS/MS analyzer and the solvent/analyte mixture is converted tovapor in the heated tubing of the interface. The analytes (i.e. vitaminD metabolites), contained in the nebulized solvent, are ionized by thecorona discharge needle of the interface, which applies a large voltageto the nebulized solvent/analyte mixture. The ions, i.e. precursor ions,pass through the orifice of the instrument and enter the firstquadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, allowingselection of ions (i.e., “precursor” and “fragment” ions) based on theirmass to charge ratio (m/z). Quadrupole 2 (Q2) is the collision cell,where ions are fragmented. The first quadrupole of the mass spectrometer(Q1) selects for molecules with the mass to charge ratios of thespecific vitamin D metabolites to be analyzed. Precursor ions with thecorrect m/z ratios of the precursor ions of specific vitamin Dmetabolites are allowed to pass into the collision chamber (Q2), whileunwanted ions with any other m/z collide with the sides of thequadrupole and are eliminated. Precursor ions entering Q2 collide withneutral Argon gas molecules and fragment. This process is calledCollision Activated Dissociation (CAD). The fragment ions generated arepassed into quadrupole 3 (Q3), where the fragment ions of the desiredvitamin D metabolites are selected while other ions are eliminated.

The methods of the invention may involve MS/MS performed in eitherpositive or negative ion mode. Using standard methods well known in theart, one of ordinary skill is capable of identifying one or morefragment ions of a particular precursor ion of a vitamin D metabolitethat can be used for selection in quadrupole 3 (Q3). Preferably, atleast one fragment ion of the method is specific for the particularvitamin D metabolite of which detection is desired. A specific fragmention for a particular vitamin D metabolite is one that will not be formedin significant amounts by other molecules with similar molecularstructures. In contrast a non-specific fragment ion is one that isformed by related molecules other than the desired analyte. Therefore,detection of non-specific fragment ions alone is not reliable fordistinguishing the desired vitamin D metabolite from other moleculesthat form the same or similar fragment ions. Specific fragment ions of aparticular vitamin D metabolite can be identified by testing variousmolecular standards (e.g. vitamin D metabolites other than themetabolite to be detected) to determine whether fragment ions formed bythe vitamin D metabolite of interest are also formed by other moleculeswith similar structures or features. In certain particularly preferredembodiments, a specific fragment ion is identified by testing at leastone molecular standard that forms a precursor ion with the same m/z asthe vitamin D metabolite to be detected.

If the precursor ion of a vitamin D metabolite of interest includes analcohol or amine group, fragment ions are commonly formed that representa dehydration or deamination of the precursor ion, respectfully. In thecase of precursor ions that include an alcohol group, such fragment ionsformed by dehydration are caused by a loss of one or more watermolecules from the precursor ion (i.e., where the difference in m/zbetween the precursor ion and fragment ion is about 18 for the loss ofone water molecule, or about 36 for the loss of two water molecules,etc.). In the case of precursor ions that include an amine group, suchfragment ions formed by deamination are caused by a loss of one or moreammonia molecules (i.e. where the difference in m/z between theprecursor ion and fragment ion is about 17 for the loss of one ammoniamolecule, or about 34 for the loss of two ammonia molecules, etc.).Likewise, precursor ions that include one or more alcohol and aminegroups commonly form fragment ions that represent the loss of one ormore water molecules and/or one or more ammonia molecules (e.g., wherethe difference in m/z between the precursor ion and fragment ion isabout 35 for the loss of one water molecule and the loss of one ammoniamolecule). Generally, the fragment ions that represent dehydrations ordeaminations of the precursor ion are not specific fragment ions for aparticular analyte. For example, MS/MS performed to detect 25OHD₂ byselecting for a precursor ion at 413 m/z (i.e. the protonated andhydrated ion) and detecting a fragment ion of 395 m/z (representing adehydration of the 413 m/z precursor ion) would not be able todistinguish 25OHD₂ from 1α(OH)D₂ which would form the same precursor andfragment ions. Therefore a 395 m/z fragment ion is not a specificfragment ion for 25OHD₂ or 1α(OH)D₂. Accordingly, in preferredembodiments of the invention, MS/MS is performed such that at least onefragment ion of a vitamin D metabolite is detected that does notrepresent only a loss of one or more water molecules and/or a loss ofone or more ammonia molecules from the precursor ion.

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, are measured and thearea or amplitude is correlated to the amount of the analyte (vitamin Dmetabolite) of interest. In certain embodiments, the area under thecurves, or amplitude of the peaks, for fragment ion(s) and/or precursorions are measured to determine the amount of a vitamin D metabolite. Asdescribed above, the relative abundance of a given ion can be convertedinto an absolute amount of the original analyte, i.e., vitamin Dmetabolite, using calibration standard curves based on peaks of one ormore ions of an internal molecular standard, such as ⁶D-25OHD₃.

In certain aspects of the invention, the quantity of various ions isdetermined by measuring the area under the curve or the amplitude of thepeak and a ratio of the quantities of the ions is calculated andmonitored (i.e. “daughter ion ratio monitoring”). In certain embodimentsof the method, the ratio(s) of the quantity of a precursor ion and thequantity of one or more fragment ions of a vitamin D metabolite can becalculated and compared to the ratio(s) of a molecular standard of thevitamin D metabolite similarly measured. In embodiments where more thanone fragment ion of a vitamin D metabolite is monitored, the ratio(s)for different fragment ions may be determined instead of, or in additionto, the ratio of the fragment ion(s) compared to the precursor ion. Inembodiments where such ratios are monitored, if there is a substantialdifference in an ion ratio in the sample as compared to the molecularstandard, it is likely that a molecule in the sample is interfering withthe results. To the contrary, if the ion ratios in the sample and themolecular standard are similar, then there is increased confidence thatthere is no interference. Accordingly, monitoring such ratios in thesamples and comparing the ratios to those of authentic molecularstandards may be used to increase the accuracy of the method.

In certain aspects of the invention, MS/MS is performed in positive ionmode with the first quadruple (Q1) tuned to select for precursor ionswith a mass charge ratio corresponding to protonated and dehydrated ionsof vitamin D metabolites. The mass/charge ratio (m/z) for the protonatedand dehydrated precursor vitamin D metabolite ions is about 383.16 m/zfor 25-hydroxyvitamin D₃ and about 395.30 m/z for 25-hydroxyvitamin D₂.In embodiments where the samples are spiked with hexadeuterated 25OHD₃(⁶D-25OHD₃) for use as an internal standard, the mass/charge ratio (m/z)of the protonated and dehydrated ⁶D-25OHD₃ precursor ion is about389.20. In certain preferred embodiments of the invention, themass/charge ratio (m/z) for the 25-hydroxyvitamin D₃ precursor ion isabout 383.16 and the m/z for at least one 25-hydroxyvitamin D₃ fragmention is about 211.35. In related embodiments, the m/z for the25-hydroxyvitamin D₂ precursor ion is about 395.30 and the25-hydroxyvitamin D₂ fragment ion(s) include one or more ions selectedfrom the group consisting of ions with mass/charge ratios (m/z) of about179.10, about 209.20 and about 251.30. In embodiments where the samplesare spiked with ⁶D-25OHD₃ for use as an internal standard themass/charge ratio (m/z) for the protonated and dehydrated ⁶D-25OHD₃precursor ion is about 389.20 and the fragment ion(s) may include afragment ion with a m/z of about 211.30.

In other aspects, MS/MS is performed in positive ion mode with the firstquadruple (Q1) tuned to select for precursor ions with a mass chargeratio corresponding to protonated and hydrated ions of vitamin Dmetabolites. The mass/charge ratio (m/z) for the protonated and hydratedprecursor vitamin D metabolite ions are about 401 m/z for25-hydroxyvitamin D₃ and about 413 m/z for 25-hydroxyvitamin D₂. Incertain preferred embodiments of the invention, the mass/charge ratio(m/z) for the 25-hydroxyvitamin D₃ precursor ion is about 401 and the25-hydroxyvitamin D₃ fragment ion(s) include one or more ions selectedfrom the group consisting of ions with mass/charge ratios (m/z) of about365 and about 383 m/z; more preferably the fragment ions include an ionwith a m/z of about 365. In related embodiments, the m/z for theprotonated and hydrated 25-hydroxyvitamin D₂ precursor ion is about 413m/z, and the 25-hydroxyvitamin D₂ fragment ion(s) include one or moreions with mass/charge ratios (m/z) of about 377 and about 395; morepreferably the fragment ions include an ion with a mass charge ratio ofabout 377.

In particularly preferred embodiments of the invention, the presence orabsence or amount of two or more vitamin D metabolites in a sample aredetected in a single assay using the above described MS/MS methods.

Mass spectrometry instruments can vary slightly in determining the massof a given analyte. Thus, the term “about” in the context of mass of anion or the m/z of an ion refers to +/−0.5 atomic mass unit.

The following examples serve to illustrate the invention. These examplesare in no way intended to limit the scope of the invention.

EXAMPLES Example 1: Determination of 25-hydroxyvitamin D₃ and25-hydroxyvitamin D₂ by LC-MS/MS

Using a Perkin-Elmer MultiProbe II (S/N 432400) robotic liquid handler,human serum samples were first extracted using a protein precipitationmethod by adding 42.5 μl of serum to 170 μl of methanol (1:4 ratio ofserum:methanol) in a 96-well plate format. For validation-relatedexperiments, the methanol was spiked with hexadeuterated 25OHD₃(⁶D-25OHD₃) as an internal standard. The 96 well plates were centrifugedto remove precipitated protein, leaving the vitamin D metabolites in thesupernatant. The supernatants were then transferred to an HPLCautosampler for loading to the LC-MS/MS analyzer.

LC-MS/MS was performed using a Thermo Finnigan LC-MS/MS analyzer (ThermoFinnigan Quantum TSQ (S/N: TQU00655)) with an atmospheric pressurechemical ionization (APCI) source as the detector. An autosampler wasused to inject 50 μL of extracted sample supernatant onto an HPLCcolumn. Liquid chromatography was performed with a Cohesive TechnologiesAria TX-4 (S/N: SJCTX409) LC system with Waters Symmetry C18 5 μm 4.6×50mm columns using 100% methanol as the mobile phase. After the analyteseluted and the detector window completed acquisition, the system waswashed with 85% Mobile phase A and then re-equilibrated with Mobilephase B for a run time of 5 minutes. Mobile phase A was 0.1% formic acidin HPLC-grade water and mobile phase B was 100% methanol.

The flow of liquid solvent exiting the HPLC column entered the heatednebulizer interface of the Thermo Finnigan LC-MS/MS analyzer. Thesolvent/analyte mixture was first converted to vapor in the heatedtubing of the interface. The analytes, contained in the nebulizedsolvent, were ionized (a positive charge added) by the corona dischargeneedle of the interface, which applies a large voltage to the nebulizedsolvent/analyte mixture. The ions pass through the orifice of theinstrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 andQ3) are mass filters, allowing selection of ions based on their mass tocharge ratio (m/z). Quadrupole 2 (Q2) is the collision cell, where ionsare fragmented.

The first quadrupole of the mass spectrometer (Q1) selected formolecules with the mass to charge ratios of (protonated and dehydrated)25OHD₂, 25OHD₃ and ⁶D-25OHD₃. Ions with these m/z ratios (see tablebelow) were allowed to pass into the collision chamber (Q2), whileunwanted ions with any other m/z collide with the sides of thequadrupole and are eliminated. Ions entering Q2 collide with neutralArgon gas molecules and fragment. The fragment ions generated are passedinto quadrupole 3 (Q3), where the fragment ions of 25OHD₂, 25OHD₃ and⁶D-25OHD₃ were selected (see table below) and other ions are eliminated.The following mass transitions were used for detection and quantitationduring validation:

TABLE 1 Mass transitions for selected vitamin D metabolites CompoundPrecursor Ion (m/z) Fragment Ions (m/z) 25OHD₂ 395.30 179.10, 251.30,209.20 25OHD₃ 383.16 211.35 ⁶D-25OHD₃ 389.20 211.30

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC methods.

Area ratios of the analyte and internal standard (Hexadeuterated25-Hydroxyvitamin D₃, ⁶D-25OHD₃) peaks were used to constructcalibration curves, which were then used to calculate analyteconcentrations. Using the calibration curves, the concentrations of25OHD₂ and 25OHD₃ were quantitated in the patient samples.

Example 2: Intra-Assay and Inter-Assay Precision

Stock solutions of 25OHD₂ and 25OHD₃ were added to pooled serum toproduce a Low Pool (20-25 ng/mL of each metabolite), a Medium Pool(45-55 ng/mL of each metabolite) and a High Pool (100-110 ng/mL). Pooledpatient serum was used for the Medium and Low Pools, and stripped serumfrom Golden West Biologicals, Product #SP1070, was used for the LowPool. Twenty aliquots from each of the Low, Medium and High Pools wereanalyzed in a single assay using the LC-MS/MS protocols described inExample 1. The following precision values were determined:

TABLE 2 Intra-Assay Variation: 25-Hydroxyvitamin D₂ (25OHD₂) Low MediumHigh 092804-L 092804-M 092804-H 1 26.5 46.4 103.3 2 23.2 51.1 96.7 323.1 52.4 107.8 4 21.6 50.3 104.5 5 26.3 47.5 96.2 6 25.1 54.4 98.5 725.9 54.6 100.0 8 21.9 50.1 110.1 9 23.4 50.8 97.6 10 23.5 53.2 105.1 1122.2 52.9 105.9 12 24.0 54.6 94.5 13 26.2 49.4 93.4 14 24.1 59.0 113.015 25.8 52.9 112.4 16 23.9 59.2 113.4 17 29.5 52.4 107.7 18 24.2 50.0115.5 19 19.8 53.5 114.9 20 26.3 60.2 126.6 Average (ng/mL) 24.3 52.7105.9 Std Dev 2.2 3.6 8.6 CV (%) 9.0 6.9 8.1

TABLE 3 Intra-Assay Variation: 25-Hydroxyvitamin D₃ (25OHD₃) Low MediumHigh 092804-L 092804-M 092804-H 1 22.7 43.6 99.1 2 22.4 45.3 93.5 3 22.450.7 98.2 4 21.0 40.1 95.9 5 21.8 41.5 82.0 6 20.8 42.2 97.4 7 22.9 50.196.0 8 19.0 42.0 106.7 9 21.8 44.2 96.6 10 23.4 49.5 94.9 11 21.8 46.597.9 12 20.7 49.9 87.1 13 25.4 44.7 85.5 14 24.5 48.0 101.5 15 25.1 45.8101.5 16 22.5 52.0 104.7 17 29.2 45.9 107.7 18 19.5 49.3 107.6 19 18.149.6 109.4 20 24.8 49.3 116.1 Average (ng/mL) 22.5 46.5 99.0 Std Dev 2.53.5 8.4 CV (%) 11.2 7.5 8.5

Each of the Low, Medium and High Pools described above were alsoanalyzed to determine inter-assay precision. Four aliquots from eachpool were analyzed over five different assays using the LC-MS/MSprotocols described in Example 1. The following precision values weredetermined:

TABLE 4 Inter-Assay Variation: 25-Hydroxyvitamin D₂ (25OHD₂) Low MediumHigh 092804-L 092804-M 092804-H 1 26.5 46.4 103.3 2 23.2 51.1 96.7 323.1 52.4 107.8 4 21.6 50.3 104.5 5 26.3 47.5 96.2 6 25.1 54.4 98.5 725.9 54.6 100.0 8 21.9 50.1 110.1 9 23.4 50.8 97.6 10 23.5 53.2 105.1 1122.2 52.9 105.9 12 24.0 54.6 94.5 13 26.2 49.4 93.4 14 24.1 59.0 113.015 25.8 52.9 112.4 16 23.9 59.2 113.4 17 29.5 52.4 107.7 18 24.2 50.0115.5 19 19.8 53.5 114.9 20 26.3 60.2 126.6 Average (ng/mL) 24.3 52.7105.9 Std Dev 2.2 3.6 8.6 CV (%) 9.0 6.9 8.1

TABLE 5 Inter-Assay Variation: 25-Hydroxyvitamin D₃ (25OHD₃) Low MediumHigh 092804-L 092804-M 092804-H 1 22.7 43.6 99.1 2 22.4 45.3 93.5 3 22.450.7 98.2 4 21.0 40.1 95.9 5 21.8 41.5 82.0 6 20.8 42.2 97.4 7 22.9 50.196.0 8 19.0 42.0 106.7 9 21.8 44.2 96.6 10 23.4 49.5 94.9 11 21.8 46.597.9 12 20.7 49.9 87.1 13 25.4 44.7 85.5 14 24.5 48.0 101.5 15 25.1 45.8101.5 16 22.5 52.0 104.7 17 29.2 45.9 107.7 18 19.5 49.3 107.6 19 18.149.6 109.4 20 24.8 49.3 116.1 Average (ng/mL) 22.5 46.5 99.0 Std Dev 2.53.5 8.4 CV (%) 11.2 7.5 8.5

Example 3: Analytical Sensitivity: Limit of Detection and Limit ofQuantitation Studies

To determine the limit of detection of the assay, blank diluent wasanalyzed 17 times within a single run using the LC-MS/MS protocolsdescribed in Example 1. The mean and standard deviation were thencalculated. The limit of detection was determined as 2 SD above the meanof the blank peak area ratio based on a back calculation of peak arearatio against the calibration curve. The limits of detection were asfollows:

-   -   25OHD₂: 3.0 ng/mL    -   25OHD₃: 3.5 ng/mL

To determine the limit of quantitation, stock solutions of 25OHD₂ and25OHD₃ were used to generate standard curves with the followingconcentrations: 0, 2, 4, 8, 16, 32, 64 and 128 ng/mL. The dilutedsamples of the standard curve were analyzed in quadruplicate over fiveassays using the LC-MS/MS assay described in Example 1. The results ofthe study were as follows:

TABLE 6 Limit of Quantitation Study Results: 25-Hydroxyvitamin D₂(25OHD₂) #1 #2 #3 #4 #5 Summary  0 ng/mL −1.2 −1.3 −0.5 −1.2 −1.1Average (ng/mL) −1.0 −1.1 −1.4 −0.9 −1.3 −0.7 Standard Deviation 0.6−1.1 −1.5 −0.8 NA −2.6 C of V (%) 59.0 0.4 −1.7 −0.6 −0.5 −0.6 Accuracy(%) N/A  2 ng/mL 3.1 2.3 2.0 2.1 3.3 Average (ng/mL) 1.9 2.1 2.7 2.1 2.41.9 Standard Deviation 0.6 1.1 1.9 1.9 0.9 1.3 C of V (%) 33.7 1.2 1.12.1 1.8 1.4 Accuracy (%) 103.9  4 ng/mL 3.5 3.9 5.0 4.5 4.8 Average(ng/mL) 3.9 4.0 3.0 3.8 3.8 4.7 Standard Deviation 0.7 3.6 2.9 2.8 3.12.0* C of V (%) 17.1 4.1 4.6 4.5 4.4 3.7 Accuracy (%) 101.7  8 ng/mL10.2 9.1 9.1 8.8 9.8 Average (ng/mL) 8.6 7.8 8.1 7.9 8.4 9.0 StandardDeviation 0.8 8.6 8.3 7.4 8.4 7.5 C of V (%) 9.3 10.2 8.4 8.0 8.1 8.6Accuracy (%) 93.2 16 ng/mL 16.0 14.8 14.4 16.7 18.3 Average (ng/mL) 16.015.5 15.6 15.3 16.7 16.8 Standard Deviation 1.1 16.6 16.7 16.8 15.8 15.2C of V (%) 7.1 14.1 17.6 16.1 16.7 14.1 Accuracy (%) 100.1 32 ng/mL 31.339.9* 29.9 33.2 32.7 Average (ng/mL) 31.8 31.7 30.5 32.4 34.0 32.5Standard Deviation 1.9 29.5 31.2 30.2 35.7 28.7 C of V (%) 6.0 32.9 34.732.4 30.8 29.2 Accuracy (%) 100.7 64 ng/mL 66.5 62.2 68.5 62.6 68.8Average (ng/mL) 64.6 66.6 67.8 67.3 58.9 61.5 Standard Deviation 3.264.4 61.4 63.7 63.5 61.3 C of V (%) 4.9 63.7 60.7 65.4 70.8 65.9Accuracy (%) 99.1 128 ng/mL  125.1 128.2 123.4 127.8 124.1 Average(ng/mL) 126.9 127.6 134.4 127.3 128.4 132.1 Standard Deviation 3.5 128.9124.5 128.5 126.5 131.7 C of V (%) 2.8 126.1 119.7 127.9 121.2 125.0Accuracy (%) 100.8

TABLE 7 Limit of Quantitation Study Results: 25-Hydroxyvitamin D₃(25OHD₃) Day #1 Day #2 Day #3 Day #4 Day #5 (11/19/04-1) (11/19/04-2)(11/22/04-1) (11/23/04-1) (11/23/04-2) Summary  0 ng/mL −0.5 −0.9 −0.30.2 −0.6 Average (ng/mL) −0.7 −0.7 −1.3 0.3 −1.1 −1.0 Standard Deviation0.6 0.0 −1.0 −1.1 −1.3 0.6 C of V (%) 86.4 −0.3 −1.5 −1.1 −0.8 −1.0Accuracy (%) N/A  2 ng/mL 2.6 1.5 2.4 2.5 1.7 Average (ng/mL) 1.9 1.70.9 3.2 2.2 1.9 Standard Deviation 0.6 1.8 1.8 2.0 1.1 2.2 C of V (%)31.4 1.3 2.4 1.5 1.1 2.5 Accuracy (%) 104.7  4 ng/mL 3.9 3.5 4.8 4.0 3.4Average (ng/mL) 3.8 3.0 4.1 3.1 3.9 3.8 Standard Deviation 0.8 4.5 3.72.9 3.5 2.4 C of V (%) 19.7 4.0 5.2 3.8 3.8 5.5 Accuracy (%) 104.1  8ng/mL 10.3 9.3 7.2 8.4 8.6 Average (ng/mL) 8.7 10.6 8.5 7.2 10.0 9.6Standard Deviation 1.1 7.4 10.3 9.0 9.4 8.4 C of V (%) 13.1 9.3 7.7 8.57.6 6.8 Accuracy (%) 91.9 16 ng/mL 15.9 15.6 16.2 18.6 17.1 Average(ng/mL) 16.0 13.8 16.3 14.0 17.2 15.3 Standard Deviation 1.4 15.6 16.115.1 18.8 16.1 C of V (%) 8.5 14.8 17.2 17.0 14.3 15.3 Accuracy (%) 99.932 ng/mL 31.1 35.8 29.6 32.8 28.0 Average (ng/mL) 31.7 30.8 29.9 31.833.0 32.8 Standard Deviation 1.9 31.6 30.9 29.5 35.7 31.2 C of V (%) 6.231.0 34.2 31.8 30.6 31.7 Accuracy (%) 101.0 64 ng/mL 65.9 64.6 64.4 64.867.8 Average (ng/mL) 64.8 67.4 62.9 62.4 60.7 57.2 Standard Deviation3.1 68.9 64.2 62.1 64.0 64.7 C of V (%) 4.8 63.2 64.2 66.8 67.7 71.1Accuracy (%) 98.8 128 ng/mL  128.9 124.5 126.4 125.3 122.8 Average(ng/mL) 127.1 129.7 135.7 128.3 125.9 128.7 Standard Deviation 4.7 125.5123.3 127.2 127.7 135.4 C of V (%) 3.7 121.3 121.8 137.8 121.4 124.1Accuracy (%) 100.7

Example 4: Assay Reportable Range and Linearity

To establish the linearity of the vitamin D metabolite LC-MS/MS assay, aMultiProbe automated liquid handler robot independently constructed twostandard curves by serially diluting a stock solution containing 128ng/mL 25OHD₂ and 128 ng/mL 25OHD₃ in 25OHD with a solution of 5% BovineSerum Albumin Fraction V dissolved in 0.01M PBS. The standard curvesamples were analyzed using the LC-MS/MS protocols described inExample 1. This process routinely produced standard curves with R²values of 0.99 or higher for each analyte for the range of 4-128 ng/mL.

To determine whether patient samples can also be diluted in a linearfashion, a total of eight samples were serially diluted with 25OHDdiluent. Two samples were patient pools (Medium and High Control Pools),three were patient samples with high 25OHD₂ values and three werepatient samples with high 25OHD₃ values. All samples were analyzed usingthe LC-MS/MS protocols described in Example 1. As shown in FIG. 1 andFIG. 2, each sample diluted in a linear fashion (R²>0.98), demonstratingthe linear range of the assay.

Additionally, solutions of 25OHD₂ and 25HOD₃ at 512 ng/mL in 5% BovineSerum Albumin Fraction V dissolved in 0.01M PBS were prepared and thenserially diluted to 8 ng/mL. Each sample was extracted and run induplicate using the LC-MS/MS protocols described in Example 1. As shownin FIG. 3, each of these curves was linear (R²>0.99).

Example 5: Accuracy of LC-MS/MS Vitamin D Assay

The stock solutions of 25OHD₂ and 25OHD₃ were quantified based upon theabsorbance of the concentrated (10-50 μg/mL) stock solutions in theultraviolet spectrum. The cis-triene chromophore present in all vitaminD compounds has a peak absorbance of 264 nm, which is dependent upon theanalyte concentration. The molar extinction coefficient of 18.3 mM⁻¹cm⁻¹was determined using purified, dessicated ergocalciferol andcholecalciferol and was used to determine the concentration of stocksolutions for 25OHD₂ and 25OHD₃.

To determine the ability to recover vitamin D metabolites from spikedserum samples, three patient pools with known levels of 25OHD₂, 25OHD₃were spiked with two levels of 25OHD₂, 25OHD₃ and both 25OHD₂ and 25OHD₃together. Each sample was extracted and run in duplicate using theLC-MS/MS protocols described in Example 1. The recovery was calculatedby dividing the expected result by the observed result.

TABLE 8 Recovery of 25-hydroxylated vitamin D metabolites from spikedsamples. 25OHD₂ 25OHD₃ 25OHD₂ 25OHD₃ (ng/mL) (ng/mL) (% Recovery) (%Recovery) Pool#1 58 51 — — Pool#1 + 20 ng/mL 25OHD₂ 75 52 104 — Pool#1 +20 ng/mL 25OHD₃ 52 68 — 105  Pool#1 + 20 ng/mL of both 72 68 109 105 Pool#1 + 50 ng/mL 25OHD₂ 104 50 105 — Pool#1 + 50 ng/mL 25OHD₃ 56 109 —93 Pool#1 + 50 ng/mL of both 104 110 104 92 Pool#2 53 47 — — Pool#2 + 20ng/mL 25OHD₂ 76 51  97 — Pool#2 + 20 ng/mL 25OHD₃ 52 66 — 101  Pool#2 +20 ng/mL of both 70 65 105 103  Pool#2 + 50 ng/mL 25OHD₂ 107 53  96 —Pool#2 + 50 ng/mL 25OHD₃ 57 109 — 88 Pool#2 + 50 ng/mL of both 100 105103 92 Pool#3 53 47 — — Pool#3 + 20 ng/mL 25OHD₂ 69 44 106 — Pool#3 + 20ng/mL 25OHD₃ 55 75 — 90 Pool#3 + 20 ng/mL of both 74 69  98 97 Pool#3 +50 ng/mL 25OHD₂ 96 48 107 — Pool#3 + 50 ng/mL 25OHD₃ 53 114 — 85Pool#3 + 50 ng/mL of both 105 113  98 85

Example 6: Comparison LC-MS/MS Vitamin D Metabolite Assay and RIAProcedures

A total of 1,057 patient samples were assayed using the LC-MS/MS methodsdescribed in Example 1 and a vitamin D radioimmunoassay commerciallyavailable from DiaSorin. FIG. 4 shows the correlation between detectionof total 25-hydroxyvitamin D using the LC-MS/MS assay and a commerciallyavailable radioimmunoassay kit; the R² value was 0.5082 with a slope of0.9684.

Example 7: Selectivity of the LC-MS/MS Assay

Samples containing various commercially available vitamin D metabolitesand analogues at a concentration of 100 ng/mL were prepared. The sampleswere extracted and run in duplicate using the LC-MS/MS methods describedin Example 1. None of the tested compounds exhibited detectable crossreactivity in the 25OHD₂ and 25OHD₃ assays.

TABLE 9 Cross-Reactivity of the LC-MS/MS method with various vitamin Danalogues and metabolites. Compound Cross- Cross- Mass ReactivityReactivity (Da) (25OHD₂) (25OHD₃) 25-Hydroxyvitamin D₂ 412 (100%) ND(25OHD₂) 25-Hydroxyvitamin D₃ 400 ND (100%) (25OHD₃) Internal Standard(⁶D-25OHD₃) 406 ND ND Vitamin D₂ (Ergocalciferol) 396 ND ND Vitamin D₃(Cholecalciferol) 384 ND ND 1α,25(OH)₂D₂ 428 ND ND 1α,25(OH)₂D₃ 416 NDND 25,26(OH)₂D₃ 416 ND ND 1α(OH)D₂ (Doxercalciferol) 412 ND ND 1α(OH)D₃(Alfacalcidiol) 400 ND ND

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

What is claimed:
 1. A method for determining an amount of a vitamin Dmetabolite in a sample by tandem mass spectrometry, comprising: (a)generating a protonated and dehydrated precursor ion of said vitamin Dmetabolite by atmospheric pressure chemical ionization (APCI) inpositive ion mode; (b) generating one or more fragment ions of saidprecursor ion; and (c) detecting an amount of one or more of said ionsgenerated in step (a) or (b) or both and relating the detected ions tothe amount of said vitamin D metabolite in said sample; wherein saidvitamin D metabolite is 25-hydroxyvitamin D₃ or 25-hydroxyvitamin D₂. 2.The method of claim 1, wherein said sample is subjected to apurification step prior to ionization step (a).
 3. The method of claim2, wherein said purification step comprises liquid chromatography. 4.The method of claim 3, wherein said liquid chromatography comprises highperformance liquid chromatography (HPLC).
 5. The method of claim 2,wherein said purification step comprises protein precipitation.
 6. Themethod of claim 5, wherein said purification step does not include highturbulence liquid chromatography (HTLC).
 7. The method of claim 2,wherein said purification step comprises chiral chromatography.
 8. Themethod of claim 1, wherein said vitamin D metabolite is25-hydroxyvitamin D₃.
 9. The method of claim 1, wherein said vitamin Dmetabolite is 25-hydroxyvitamin D₂.
 10. A method for determining anamount of 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂ in a sample in asingle assay, said method comprising: (a) ionizing by atmosphericpressure chemical ionization (APCI) in positive ion mode said25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂ to generate protonated anddehydrated precursor ions specific for each of said 25-hydroxyvitamin D₃and 25-hydroxyvitamin D₂; (b) generating one or more fragment ions ofeach of said precursor ions; and (c) detecting an amount of one or moreof said ions generated in step (a) or (b) or both and relating thedetected ions to the amount of 25-hydroxyvitamin D₃ and25-hydroxyvitamin D₂ in said sample.
 11. The method of claim 10, whereinsaid sample is subjected to a purification step prior to said ionizationstep (a).
 12. The method of claim 11, wherein said purification stepcomprises liquid chromatography.
 13. The method of claim 12, whereinsaid liquid chromatography comprises high performance liquidchromatography (HPLC).
 14. The method of claim 11, wherein saidpurification step comprises protein precipitation and does not includehigh turbulence liquid chromatography.
 15. The method of claim 10,wherein said sample comprises a biological sample.
 16. The method ofclaim 10, wherein said sample comprises plasma or serum.